Production and utilization of microbial fructans by enzymatic transformation of sucrose are an important object for the sugar, food, and other industries. Bacterial fructosyltransferases (EC 2.4.1.10) catalyze the synthesis of oligo- and/or polyfructans by transferring fructosyl moieties from sucrose-containing saccharides to acceptor molecules. Different compounds can be used as acceptors which allows the enzymatic production of nondigestible homo- and heterooligosaccharides with beneficial effects on humans and animals.
Most of the bacterial fructosyltransferases characterized so far are levansucrases (Cote, G. L. and Ahlgran, J. A., In Science and Technology of fructans. Metabolism in microorganisms, Part I: Levan and levansucrase. CRC Press, 1993). All levansucrases catalyze the transfructosylation reaction from sucrose to a variety of acceptors such as water (sucrose hydrolysis), glucose (exchange reaction), fructose (chain elongation) and sucrose (synthesis of oligosaccharides). However, differences have been noted between these enzymes pertaining to the relative efficiency of each reaction which leads to the accumulation of oligofructans of different polymerization degree.
Several bacteria and fungi have been identified to develop transfructosylation reactions from sucrose (for review see Cote, G. L. and Ahlgran, J. A., In Science and Technology of fructans. Metabolism in microorganisms, Part I: Levan and levansucrase. CRC Press, 1993). Fructosyltransferase genes have been isolated from Bacillus subtilis (European patent application EP 0117823 A1 840905), Bacillus amyloliquefaciens (Tang, L. B. et al., Gene 96, 89-93, 1990), Streptococcus mutans (Shiroza, T. et al., J. Bacteriol. 170, No. 2, 810-816, 1988), Streptococcus salivarius (Rathsam, C. et al., J. Bacteriol. 175, No. 14, 4520-4527, 1993), Zymomonas mobilis (Ki-Bang Song et al., Biochim. Biophys. Acta 1173, 320-324, 1993) and Erwinia amylovora (Geier, G. et al., Physiological and Molecular Plant Pathology 42, 387-404, 1993). Low homology is found among the deduced amino acid sequences of fructosyltransferases isolated from different bacterial genera.
The transfructosylation system of the genus Bacillus (Gram-positive bacteria) has been well characterized. The Bacillus subtilis levansucrase is an inducible exoenzyme which catalyzes the formation of high weight polymer without accumulation of transient oligofructans of low polymerization degree. Recombinant levansucrases of Bacillus subtilis have been obtained in genetically manipulated hosts such as bacteria (Philippe, G. J. Bacteriol. 153, No. 3, 1424-1431, 1983), yeast (Scotti, P. A. et al., Yeast 10, No. 1, 29-38, 1994) and plants (Ebskamp, M. J. M. et al., Biotechnology 12, 272-275, 1994).
Acetobacter diazotrophicus is the most recently identified species of the genus Acetobacter (Gillis et al., Int. J. Sist. Bacteriol. 39, 361-364, 1989). The cells are Gram-negative, N.sub.2 -fixing, acid-tolerant, microaerobic, straight rods with rounded ends, about 0.7 to 0.9 by .+-.2 .mu.m, motile by lateral or peritrichous flagella. The bacteria are non-pathogenic and further distinguished by their ability to establish beneficial association with sugarcane. However, the molecular biology of the bacterium has been poorly investigated.